Francisco J. Enguita

Francisco J. Enguita
University of Lisbon | UL · Institute of Molecular Medicine

77.15
 · 
B.Pharm., Ph.D.
About
531
Research items
108,006
Reads
1,332
Citations
Introduction
Our lab is interested in non-coding RNAs and human disease, using a multi-disciplinary approach that combines cell, molecular and structural biology. We are willing to collaborate with dynamic people not afraid of using and implementing new methodologies for integration of biological data in several contexts, including regulatory networks based on non-coding RNAs at the cell and organism level.
Research Experience
May 2006
Faculdade de Medicina, Universidade de Lisboa
Position
  • Senior Scientist - Principal Investigator
Description
  • Molecular biology of non-coding RNA regulatory action in human disease. Biomarkers of disease based on ncRNAs. Cardiovascular diseases. Rare genetic diseases. Non-coding RNA as inter-kingdom communicators. Non-coding RNAs involved in infection.
Jan 2002 - Mar 2006
Universidade NOVA de Lisboa
Position
  • Group Leader
Description
  • Structural studies on proteins involved in bacterial attachment to host-cells
Jan 1999 - Dec 2001
Universidade NOVA de Lisboa
Position
  • EMBO long-term fellowship
Description
  • Post-doctoral training on X-ray crystallography and protein structure
Education
Oct 1992 - Oct 1998
Universidad de Salamanca
Field of study
  • Microbiology and Genetics
Jun 1991 - May 1992
University of Granada
Field of study
  • Microbiology
Oct 1986 - Jun 1991
University of Granada
Field of study
  • Pharmaceutical Sciences
Network
Cited By
Followers
Following
Projects
Projects (9)
Project
The Cardiomics laboratory is applying a multi-disciplinary approach using structural biology, biochemistry, genetics and bioinformatics to unravel the role of non-coding RNAs in cardiovascular diseases. More specifically, the Cardiomics laboratory is currently investigating the use of circulating miRNAs as biomarkers of cardiac diseases including infarction and atrial fibrillation, the physiological role of miRNAs in rare diseases such as Fabry’s disease, and the involvement of non-coding RNAs as regulators of heart misfunction.
Project
Catalysing transcriptomics research in cardiovascular disease is a COST Action that aims to create an interdisciplinary network to accelerate the understanding of transcriptomics in cardiovascular disease and further the translation of experimental data into usable applications to improve personalized medicine in this field. http://www.cardiorna.eu
Project
Research project objectives The project is proposed to explain role of specific circulating microparticles as conveyors in trafficking bio-active molecules in type 2 diabetic patients with risk of diabetic retinopathy. The aim of this project is to describe mechanisms regulating capillary changes, arteriovenous shunts formation and neovascularization in a model in vitro including role of microparticles of different origin (platelets, endothelium, beta-cells, retinal cells) and their formation (induced by hyperglycemia or aging). Possible role of miRNA as a modulator of these processes (switching on/off mechanism on the molecular level) is proposed. Increased number of microparticles with respect to glucose concentrations after both in vivo (patients’ plasma) and in vitro (cell culture) is expected.
Research
Research items (531)
Article
Full-text available
Background: MicroRNAs (miRNAs) are key players in cardiovascular development and disease. However, not only miRNAs of a cardiac origin have a critical role in heart function. Recent studies have demonstrated that miR-122-5p, a hepatic miRNA, increases in the bloodstream during ischemic cardiogenic shock and it is upregulated in the infarcted myoca...
Article
Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by a single gene mutation, a reciprocal translocation that originates the Bcr-Abl gene with constitutive tyrosine kinase activity. As a monogenic disease, it is an optimum target for RNA silencing therapy. We developed a siRNA-based therapeutic approach in which the siRNA is del...
Article
The centrality of RNA within the biological world is an irrefutable fact that currently attracts increasing attention from the scientific community. The panoply of functional RNAs requires the existence of specific biological caretakers, RNA helicases, devoted to maintain the proper folding of those molecules, resolving unstable structures. However...
Article
Full-text available
Vascular dysfunction is a severe complication which can cause organ ischemia and damage during acute pancreatitis (AP). Laboratory assessment of AP is based on several routine parameters and does not reflect endothelial dysfunction or organ injury. Recently, small non-protein-coding RNAs (miRNAs) have been introduced to laboratory diagnostics as ne...
Article
Full-text available
Dengue, West Nile and Zika, closely related viruses of the Flaviviridae family, are an increasing global threat, due to the expansion of their mosquito vectors. They present a very similar viral particle with an outer lipid bilayer containing two viral proteins and, within it, the nucleocapsid core. This core is composed by the viral RNA complexed...
Answer
Hi there
In the meantime I discovered this tool which allow you to setup a scene with a PDB file, customize it to highlight ligands, domains, etc, and export to a URL to (for instance) share with your colleagues or students.
Take a look
Best
Answer
I executed your command, and everything is the same. When I run the script either as a self-executable or by calling Python before.. the same errors appeared
Answer
I tried to run the Python script but I got this error:
/usr/bin/env: ‘python\r’: No such file or directory
What I am doing wrong?. I put the script in the folder where the list of *.PDBQT files are generated.
If I try to run the script by calling Python first I got this other error:
File "vina_screen_get_top.py", line 10
print 'Found', len(file_names), 'pdbqt files'
^
SyntaxError: invalid syntax
Can you help me please?. Best and thanks
Question
Dear All
I did a virtual drug screening with Autodock-Vina, and I have generated a list of log files (one for each ligand analyzed) and solutions in PDBQT format. Do you have some script or method to rank for the best solution (compound) from such a list of log or PDBQT files from Vina?
Thanks in advance.
Article
Background: Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia in western countries. The factors governing the progression of AF to a permanent chronic condition are still not well characterized. Among epigenetic factors, non-coding RNAs (ncRNAs) such as miRNAs and lncRNAs have been recently described as important players involved in...
Question
I have a protein-drug complex obtained by virtual drug screening. How can I refine the coordinates of the whole complex?. Is energy minimization the best protocol to be applied?
Thanks in advance. Waiting for your valuable opinions.
Question
I am looking for an alternative to the former and discontinued Autodesk Molecule Viewer platform. I would need to make molecular representations available on the web for teaching purposes.
In the Molecule Viewer, it was possible to create scenes that were available on the web by a URL address, which was automatically created by the application. The user was able to open the URL with a net browser and to interact with the scene (rotate, zoom, change representation type, etc).
Any suggestions?. Thanks in advance
Preprint
Dengue virus (DENV) and Zika virus (ZIKV) are both positive sense single-stranded RNA viruses. They are packaged within the virion with a capsid (C) protein to form the nucleocapsid. Based on cryo-electron microscopy imaging, the nucleocapsid has been described as lacking symmetry, whilst there is distinguishable separation of the C proteins from t...
Article
Full-text available
Cardiovascular disease (CVD) remains the leading cause of death worldwide and, despite continuous advances, better diagnostic and prognostic tools, as well as therapy, are needed. The human transcriptome, which is the set of all RNA produced in a cell, is much more complex than previously thought and the lack of dialogue between researchers and ind...
Article
Full-text available
The fermentation process is widely used in the industry for bioethanol production. Even though it is widely used, microbial contamination is unpredictable and difficult to control. The problem of reduced productivity is directly linked to competition for nutrients during contamination. Yeasts representing the Candida species are frequently isolated...
Question
Dear All
I am looking forward to treat my total RNA samples with RNAse R. Any suggestions about which supplier do you prefer for purchasing it?.
Thanks. Best
Answer
Ana
Looks that you want to insert something in the middle of a gene to knock-out it. If yes.. a single gRNA should be enough, but you need to design the gRNA close to the middle region of the recombination area.
Of course 2 gRNAs will give you better results, as far as you can eliminate a big fragment of DNA.
For transformation of the gRNAs tandem systems are OK, but if the transformation efficiency of your cells is high you can also think about using the synthetic gRNA directly, without the need of expressing it.
Hope it helps. Best
Article
Full-text available
The reduced expression of solute carrier family 2, facilitated glucose transporter member 4 (GLUT4) and hexokinase-2 (HK2) in skeletal muscle participates in insulin resistance of diabetes mellitus (DM). MicroRNAs (miRNAs) have emerged as important modulators of mRNA/protein expression, but their role in DM is unclear. We investigated miRNAs hypoth...
Article
Full-text available
The reduced expression of solute carrier family 2, facilitated glucose transporter member 4 (GLUT4) and hexokinase-2 (HK2) in skeletal muscle participates in insulin resistance of diabetes mellitus (DM). MicroRNAs (miRNAs) have emerged as important modulators of mRNA/protein expression, but their role in DM is unclear. We investigated miRNAs hypoth...
Cover Page
Full-text available
Front Cover: Circulating ectosomes: Determination of angiogenic microRNAs in type 2 diabetes
Answer
Hi Agnes
I should advice you to use the "Maniatis" protocol.
Standard Washing:
- wash 2x for 15 min in 50 ml of 2x SSC, 0.1 % SDS at room
temperature
- wash 2x for 15 min in 50 ml of 0.5x SSC, 0.1 % SDS at 55 ̊C
(prewarmed)
or
for higher stringency:
wash 2x for 15 min in 50 ml of 0.1x SSC, 0.1 % SDS at 60 ̊C
(prewarmed)
Hope it helps
Best
Paco
Question
Dear All
Do you have experience in methods for the over-expression of selected circular RNAs in human or mouse cells?... If yes... which strategy did you use?... which vectors and promoters?...
Thanks in advance. Best
Paco
Cover Page
Full-text available
Schematic representation of circulating miRNAs detected in plasma or serum, and characterized as biomarkers of at least two cardiovascular diseases
Article
Ectosomes (Ects) are a subpopulation of extracellular vesicles formed by the process of plasma membrane shedding. In the present study, we profiled ectosome-specific microRNAs (miRNAs) in patients with type 2 diabetes mellitus (T2DM) and analyzed their pro- and anti-angiogenic potential. Methods: We used different approaches for detecting and enume...
Question
Hi there
I would like to know and to get your feedback about you favourite Gene-enrichment analysis software based in a graphical environment, preferably on-line.
Please give me your feedback about what you like most !... Thanks
All the best
Paco
Answer
Hi Daniel
Thanks for the answer... Do you know the cost per sample to run those Starter packs for miRNAs?...
Question
Dear All
I wanted to give a try to Nanostring system to detect extracellular miRNAs in a screening-based approach. I am wondering if some of you have experience with Nanostring in the detection of miRNAs from fluids.
How is the performance of the system used with low-quality RNA samples?
Waiting for your feedback. Thanks.
Answer
Hi David
Difficult question. There is no specific rule to cover the whole circRNA transcriptome, but of course the higher redundancy the better. You can also try to do ribo- and polyA- RNA to specifically enrich those transcripts without polyAs and increase the yield of detection for circRNAs.
Best. Paco
Answer
Hi Ketharini
RIN is an indication of the integrity of RNA. It is important for quality control of every sample of total RNA (where you can localize the ribosomal RNAs). Of course that a bad RIN in a sample, will indicate degradation, and presence of small RNA fragments that will be subsequently purified and sequenced in the small RNA-seq experiment.
So, the better the RIN, the more guarantee you have to see only small RNAs in your sequencing and not degradation products.
Best
Answer
Dear Eric
Small ncRNAs have tendency to be relatively conserved in sequence among organisms of the same groups, lets say mammals. However, you can always find small ncRNAs that are specific from sub-grups, for instance primates. Check this website, that pinpoint miRNA orthologs for several organisms:
In the case of long ncRNAs (lncRNAs), the scenario cannot be consider so optimistic. There are huge amounts of specific lncRNAs that you can find in humans and not in mouse. In the opposite direction, some lncRNAs present in mouse cannot be found in humans (just by sequence conservation), and you would need to do functional assays to demonstrate their existence.
There is a very cool case to illustrate how careful you must be in the case of lncRNAs. There is a lncRNA called "Braveheart" which was initially characterized in mouse as a regulator for the proper recruitment of cardiomyocyte precursors and formation of a functional heart. This lncRNA is absent in humans, and we have other ways to do the same things with different lncRNAs.
Best
Answer
Hi there
Please check if you find your mutation in some of the databases described in this protocol
If you cannot find it and you are dealing with a new SNP, I am afraid that you should perform a dirty analysis with miRANda or any other basic tool, using the sequence of the miRNA against the given genome. You need to install miRanda in a Linux box, and download your genome to interrogate it.
Best
Answer
Hi there
CRISPR is a primitive immune system developed by bacteria to defend themselves against viruses. The idea is simple: when infected by viruses, bacteria are able to integrate portions of the genome of the viruses within their own genomes. In the case of a recurent infection of the same virus, the inserted portions of virus genomes will be transcribed to produce a RNA molecule, that will be used by the bacteria to target and degrade the infecting genome. The responsible for the degradation of the viral genome is a protein called Cas, which will recognize the virus DNA together with the generated RNA, which will bind to the virus genome by Watson-Crick complementarity.
The funny thing about this system is that if you are able to design a RNA complementary to a given DNA, Cas protein will cleave the target DNA. For this reason it is used in "genome editing" protocols. It works wonderful in any kind of cell, human, plants, etc. Cleaving a genomic DNA in a specific region could facilitate deletions or homologous recombinations with a high efficiency. So, you can virtually "copy and paste" anything in whatever location of a specific genome.
Hope it helps. Best
Article
Pervasive transcription of the human genome is responsible for the production of a myriad of non-coding RNA molecules (ncRNAs) some of them with regulatory functions. The pivotal role of ncRNAs in cardiovascular biology has been unveiled in the last decade, starting from the characterization of the involvement of micro-RNAs in cardiovascular develo...
Answer
Hi David
If you have differences in splicing patterns depending on the collection time of the samples, be sure that you will also see differences in the circRNA expression pattern (quality and quantity), since these species are depending on non-canonical splicing.
Best
Answer
Hi
You can use KEGG database or Biocarta to extract the list of genes related with a pathway or process. I particularly like this application called Harmonizome, a curated system for retrieval of genes related with specific functions. You can search your Biocarta pathway and obtain your list of genes at
Hope it helps. Best
Paco
Article
γδ T cells are major providers of proinflammatory cytokines. They are preprogrammed in the mouse thymus into distinct subsets producing either interleukin-17 (IL-17) or interferon-γ (IFN-γ), which segregate with CD27 expression. In the periphery, CD27⁻ γδ (γδ27⁻) T cells can be induced under inflammatory conditions to coexpress IL-17 and IFN-γ; the...
Answer
Hi Iason
Try to include 6-12% DMSO in your reaction mix to increase astringency. Your primers look in the lower limit of GC content (around 40%). If DMSO fails, you can think in re-designing primers with higher GC
Best
Paco
Answer
Hi Mona
Just to complement the already posted info.
1.- For circRNAs you should use random primers for RT reaction.
2.- For miRNAs the majority of the suppliers use a different approach. First, a polyadenilate kinase is used to synthesize a poly-A stretch in your RNA, and then the RT reaction is primed by an oligo-dT.
Hope it helps
Best of luck
Paco
Article
Gamma-delta-T cells are major providers of proinflammatory cytokines. They are preprogrammed in the mouse thymus into distinct subsets producing either interleukin-17 (IL-17) or interferon-gamma (IFN-gamma), which segregate with CD27 expression. In the periphery, CD27− gamma-delta (gamma-delta-27−) T cells can be induced under inflammatory conditio...
Answer
Dear Kathleen
You are probably facing a problem of efficiency in the transfection as you suspected. For such a long UTR I would advice you to chop it out into pieces including only your target sites. It is somehow non-physiological, but you should get better transfection efficiency.
Luciferase assays are very artificial, and I am not a particular fan of them. Remember that you will change the physiological environment of your UTR probably using a different cell line, and also removing the rest of the mRNA.
I would rather prefer to use AgoIP analysis to test for enrichment of your targets.
Best
Answer
Hi Andrea
Totally right... labelling and ID of the plates is a chaos. I forgot to tell these people about.
Answer
The Qiagen's official answer to my complaints... Still not convincing to me. Anyhow... look and judge by yourselves.
Qiagen: Dear Francisco J. Enguita,
thank you very much for your prompt reply and summarizing the main issues. let me try to comment the different points and explain that you have faced a strange and non-acceptable quality of the plates. In addition, please find a letter describing the changes QIAGEN made for producing the miRCURY PCR plates to follow the QIAGEN production standards.
FE: 1.- Primers are coming resuspended in a pink liquid, instead of being liophylized as Exiqon used to do. The volume of the pink liquid where the primers are resuspended is not known (at least I have no information about). Dilution effects over the Sybrgreen mix should be taken into account.
Qiagen: Resuspended primers (liquid)  in the plate is of course a no go and a strange observation. We produce the plates with primers dried down and they always sit on the bottom of the wells. So, there is no pink liquid we resuspend the primers in. This observation is strange and was forwarded with your images to production in the US. We have never had such issue within many years of producing such plates. We can tell you that QIAGEN is producing such spotted plates with PCR Primers for more years and higher quantities than Exiqon did. Therefore, your observation is strange and a no go! QIAGEN is spotting the primers with a red dye (dried down) to visualize where the dried down primer pair sits for better and easier resuspension at the customer. We have of course tested extensively that the dye does not interfere with SYBR Green, EVA Green, any Probe label etc. We use the dye for many years in the plates without any complaint. However, as corrective action production tested the plate drying procedures again and prolonged the drying time for miRCURY plates already last year. It seems you have received one of the first batches which were produced. We are very sorry for that.
FE: 2.- We determined that Cts from Spike (UniSp6) are around 2 cycles above the "typical" Exiqon standards in the same conditions.
Qiagen: This issue we have heard from other customers as well and was cycler company depended and not observed during test for product transfer from Exiqon to QIAGEN. We corrected this and now Cqs are the same again.
FE: 3.- The most serious problem is that plates are not individually sealed, rather wrapped in a bag. Despite of the condensation issues that apparently only affected my plates in the whole world, how can you guarantee that there is no cross-contamination between primers in different wells if the plates are not sealed.
Qiagen: As mentioned above QIAGEN changed the plate production to the standard QIAGEN plate production, which is without sealing the plate. This might sound strange, but the production is safe and we produced many more plate that Exiqon this way and shipped worldwide without contamination or complaint issue. However, I understand that especially with your observed red liquid in the well such production looks like a fundamental problem. As the primers a dried down at the bottom and visible with the red dye contamination can be excluded. Here many tests have been performed in the past, of course. We are very sorry about the trouble and issues you have faced because of our manufacturing transfer and have not been inforemd in advance about the product updates. I would like to organize a replacement shipment (if you agree) that you can see that the quality of unsealed plates is the same as with Exiqon.
Answer
Hi Peter
If you have a preparation of total RNA, Qubit will quantify the total amount of RNA, not a specific population as the small RNAs. On the other hand, if you have a size-fractionated preparation of RNAs (as those obtained by miRVana fractionation protocol), you will be quantiying this population in Qubit.
Best
Answer
The saga continues
********
Dear Francisco
I just tried to reach you on the phone. We will of course issue a replacement for you, I will kindly ask you to send me a copy of your order so I can make sure the correct products are included in the replacement. Furthermore, I will again escalate your case to the responsible people, so we can find a long-term solution which is acceptable to the customers. I hope I can give you an update on the matter during next week, when the Product Manager is back in the office. We do take this very seriously, the quality of our products should never affect your research.
You are welcome to email or call me with any questions or comments, my phone number is found below.
Answer
Dear ALL and question followers... I have complained to the customer support email from Exiqon and I received this answer from them. I will wait for further developments and I will let you know.
Best. Paco
***********************
Dear Francisco
Thank you for your mail. I can fully understand why you are not satisfied with the packaging solution. I am working on a solution for you, I just wanted you to know that you have not been forgotten. I will come back with an update as soon as I know more. You can reach me directly at this mail address and the phone numbers below, and my colleagues are available at gs.emea@qiagen.com
Kind regards,
Answer
Dear Andrea and followers
We have finished using the plates for a screening, and the Ct values are around 1-2 cycles bigger that those observed with the Exiqon plates. I do not know if this is because of the design of the plates.
Please let me know your feedback about.
I think that we need to contact directly those people but for me is really difficult because they have local "dealers".
Best
Paco
Answer
Hi Petra and followers of the question
I just performed a live "unboxing" of one of the screening plates that we talked about. Check out the aspect of the condensation droplets and the lack of sealing.
Best. Paco
Answer
Thanks Petra
I am afraid the customer service of Qiagen in Portugal is not as good as you imagine.
Best
Answer
Petra
It looks that the plates are supplied like that now. I got used to the old setup from Exiqon where primers were liophilized and dry. Don't you think that this is not the right way to sell such kind of screenings?
Thanks
Paco
Question
Dear All
I had used for a while the Exiqon miRNAnome panels based on low density qPCR arrays. However, since the begginning of this 2018, Exiqon has joined Qiagen.
I ordered the same screenings from Qiagen-Exiqon, and now the primers are not liophilized, but in an aqueous solution, and also the plates are not sealed. The lack of sealing of the plates generates condensation in the bag that contained them, and I am afraid that this could interfer the proper standarization and comparison of samples. This is a real drawback in quality if we compared with the elegant, liophilized and sealed Exiqon plates, which I miss so much.
Do you have such experience?. What's your opinion about the new Exiqon-Qiagen joint venture?. Is is worthwile to continue using such screenings or should I move to the Taqman?
Thanks in advance. Best
Paco
Article
Micro-RNAs (miRNAs) are small non-coding RNAs that act as negative regulators of the genomic output. Their intrinsic importance within cell biology and human disease is well known. Their mechanism of action based on the base pairing binding to their cognate targets have helped the development not only of many computer applications for the predictio...
Poster
Full-text available
Ectosomes (ECTS) belong to a large family of membrane vesicles or EVs, that are released from cells. They constitute a heterogeneous population of vesicles with diameters in the range between 0.1 to 1 micron, which retain many features of parental cells including surface antigens and receptors. In the blood, endothelial cell- and platelet-derived E...
Answer
Hi Karel
There are some tools around to check for alternative splicing in your gene, but of course it depends on the organism that you are studying. Everything is smooth for human and model organisms, but for exotic ones the problem will be worst. Check this out:
Best. Paco
Answer
Hi there
Please check Feng Zhang's web page. It is full of tips and tricks about how to use CRISPR system in general purpose applications.
Hope it helps. Best
Paco
Answer
Hi there
Try to reduce the stringency of the buffer. I would recommend you to use DMSO, in a concentration range between 6-12 %.
Hope it helps
Best
Answer
Hi Emerson
Codon optimization is always a good practice when you are dealing with such amount or rare codon species. So, I will definetively advice you to use codon optimization. Many companies that do gene synthesis also perform codon optimization by propietary algorithms (See for instance Genescript: https://www.genscript.com/codon-opt.html). However the application that you cited is very good and reliable.
Many other bottlenecks can appear in your protein expression experiments, and it is not so easy to describe all of them. If you have access to several E.coli strains, do a expression screening in all of them and check how your protein behaves. If everything goes wrong, try to low the expression temperature (to 20-25 ºC) and do longer expressions (10h to ON). Also it is advisable not to use very high IPTG concentrations for induction. It is amazing but tac-lac promoters are almost saturated with 0.2 mM IPTG, and you will easily see "good" papers claiming that they induced at 0.5-1 mM. IPTG is toxic for the cells, and at those concentrations you can see nasty effects.
Other options that you can explore (specially if you are as lazy as I am), is the use of autoinduction media.
Hope it helps. Best
Paco
Answer
Hi there
I agree with Albert. My choice would be to use a capillary electrophoresis system (Bioanalyzer or similar). For microarray analysis you normally will start with total RNA preparations, and calculate the total RNA concentration.
I do not think that you should need to quantify the amount of miRNAs. Moreover, the quantification of total miRNA amounts is very difficult because they will appear as a low-molecular weight peak, which is also contaminated with tRNAs and other small RNAs.
Best
Article
Full-text available
Background: Circulating microRNAs (miRs) levels are potentially important diagnostic and prognostic biomarkers in acute coronary syndrome (ACS) or cerebral ischemic event (CIE) resulting from internal carotid artery stenosis (ICAS). Aim: This 4-year prospective study aimed to compare the levels of circulating miRs in ACS vs CIE patients, and inv...
Article
Full-text available
The causative agent of malaria, Plasmodium, replicates inside a membrane-bound parasitophorous vacuole (PV), which shields this intracellular parasite from the cytosol of the host cell (1) . One common threat for intracellular pathogens is the homeostatic process of autophagy, through which cells capture unwanted intracellular material for lysosoma...
Article
Introduction Circulating microRNAs (miRNAs) levels are potentially important biomarkers and therapeutic targets of cerebral ischemic event (CIE) in patients with internal carotid artery stenosis (ICAS). Aim This prospective study investigated associations between circulating miRNAs and symptomatic and asymptomatic ICAS, carotid plaque morphology a...
Answer
Hi Davide
Sorry for my silence. You were right. I installed brand new Anaconda and everything runs smoothly. I am still quite bit puzzled about how to interpret the results, specially the clustering. Do you have any manual, or tutorial where I can find more info about?.. Thanks again for such a nice piece of work.
Best. Paco
Answer
Hi Davide
I ran conan, but I got this weird error at the end of the run, when the software was trying to do clustering. I am enclosing my input file for you to check. Do I need to install anything else in Numpy?. Thanks in advance.
***********************
mkdir: created directory ‘cluster_res’
Attempting k-medoids based on interaction lifetimes...
mkdir: created directory ‘cluster_res/lifetime’
Traceback (most recent call last):
File "../../conan-master/conan.py", line 1921, in <module>
call_hierarchical("cluster_res/lifetime", vectors, trunc, trunc_inter, life_cond, xres, ks_res, "lifetime", None, 'res', xterm, xres, yterm, yres, asymm, matrices)
File "../../conan-master/conan.py", line 1332, in call_hierarchical
Z = linkage(condensed_dist, 'ward')
File "/usr/lib/python3/dist-packages/scipy/cluster/hierarchy.py", line 627, in linkage
raise ValueError("Valid methods when the raw observations are "
ValueError: Valid methods when the raw observations are omitted are 'single', 'complete', 'weighted', and 'average'.
***********************
Cheers
Paco
Answer
Hi Davide... Superb!!... I will check it for sure and let you know. Thanks
Answer
Dear Son
Ok.. If I run the script I convert the XPM to a text matrix. How to run the script?. After that how to do a comparison between two matrices?
Thanks in advance
Question
Hi there
I got several contact matrices in XPM format generated by GROMACS trajectories for a series of protein mutants. How to compare them to extract the differences between atom contacts in each mutant?
Thanks in advance.
Answer
Hi Ammar
For protein visualization I would recommend Pymol. There is a free version of the software, however the most updated is payed.
For protein tertiary structure prediction there are a bunch of web servers that would scan for your sequence to predict the 3D structure if they find something similar in the PDB.
Basic and based on homology modelling: Phyre2
More advanced and combining homology modelling and threading: I-Tasser
Hope it helps. Best
Answer
Dear Ilaria
I am sorry to dissapoint you but I do not think that it makes any sense to think about RIN in urine or in other biofluids, unless they are heavily contaminated with cells. The RIN is calculated by the ratio between the abundance of ribosomal RNAs (big/small), so if you are working with a fluid (plasma, urine, tears, saliva, etc...), I would not expect to have ribosomal RNA.
Best
Paco
Answer
Hi Marina
Just to add some info to the previous answer, if you work with a miRNA screening with 30 miRNAs or more, give a try to use the global normalization approach. It works fine for me.
I usually employ Data Assist software. It is quite old-fashion software but it is able to import tabular Ct data from many machines.
Best
Paco
Answer
Hi Soudabeh
Interesting question. In fact 3D structure of mRNA transcripts can affect gene expression by many mechanisms. The most obvious is the sterical effect that it can have in ribossome translation and dynamics, but also you have to take into account the specific 3D structures in non-coding regions, namely 5' and 3' ends that could enhance or prevent the binding of regulatory proteins or non-coding RNAs.
In the particular case of non-coding RNAs, and specifically miRNAs, there are very elegant demonstrations of the role of mRNA structure in the regulatory activity of these non-coding RNAs even in the 5'. See for illustration the following papers:
Hope it helps. Best
Paco
Article
Full-text available
Lactadherin is a small (53-66kDa) multifunctional glycoprotein belonging to the secreted extracellular matrix protein family. It has a multi-domain structure and is involved in many biological and physiological processes, including phagocytosis, angiogenesis, atherosclerosis, tissue remodeling, and haemostasis regulation. Lactadherin binds phosphat...
Answer
Hi Gary
For high GC-containing sequences I would advice you to use DMSO as an additive of your PCR reaction to increase stringency. Typical concentrations for a starting screening will be between 6 and 12 % of the final PCR reaction volume.
Good luck. Best
Paco
Answer
Hi there
I agree with Matteo. In fact sequence alteration and sequence variants appeared to be used as synonyms in many databases. If your work is related with the medical field you must consider also the concept "mutation", which is always related with a pathology (different with the mutation concept in general biology).
Best.
Paco
Answer
Hi there
Following the miRBase criteria, a real miRNA should be generated by the expression of pri-miRNA transcripts that will produce the characteristic hairpin-loop structure in the corresponding precursor-miRNA. After that you must consider the detection of the mature species in next generation sequencing experiments (or others).
There are many examples of miRNA-like sequences, many of them coming from the degradation of other RNAs, like tRNAs.
Hope it helps. Best
Paco
Answer
Hi Ege
We are routinely working with Genecore at EMBL Heidelberg. They are excellent professionals and you can easily interact with them. Prices are on the academic level, but depends of course on your experiment. Please take a look at their webpage:
Best luck. Paco
Answer
Hi Peter
Look at this in our miRNAtools3 webpage. You have a few tools to predict de novo putative miRNA-producing hairpins. Be cautious because some of them are quite outdated.
Best luck. Cheers
Paco
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Hi Matilde
In principle the answer is YES... they will share the same family of targets, however their binding affinities to the target could be different, and consequently the expected regultory effect.
Best
Paco
Answer
Hi Harris
In fact isolation of RNA from serum/plasma is a tricky bussiness. I am pretty familiar with Exiqon biofluids system and works smoothly in typical samples, unless they are hemolyzed. The aproximate yields vary from 10-50 ng/ul. IMPORTANT: You should use the pico-RNA chip in Bioanalyzer to see something, otherwise you will be in the detection limits of the system.
From CSF, and using the same kit, the yields use to be smaller (around 10 ng/ul). However under these levels, quantification is always very difficult and the results must be taken with caution.
Sometimes, you can have a decreased extraction yield due to insoluble material in your sample. I normally centrifugue the fluid (serum/plasma/CSF) at 10,000 x g during 10 minutes, before the extraction protocol.
Best of luck
Paco
Answer
Hi Qaisar
Not too much help about R-based software from this side. If you want something easy to analyze your microarray data, give a try to AltAnalyze (http://altanalyze.org/). It works on Windoze and Linux and uses pretty much the same algorithms as R for normalization and differential expression.
Best or luck
Answer
Hi Aldo
Besides Sheerien's post, you probably might give a try to LNA Gapmers. Mechanism of knocking-down is slightly different because it may involve RNAse H, but they are pretty good in silencing ncRNAs. I never tried them in ncRNA regions of coding transcripts, but the LNA will ensure a more specific and tighter binding to the target than the classical RNAi oligo.
Gapmers were sold by Exiqon. Unfortunately Exiqon has been purchased by Qiagen, and now the orders are pretty much more difficult than before.
Best
Answer
Hi Nikiforos
I am pretty familiar with Exiqon biofluids kits and I have to tell you that I never used MS2 as carrier. You basically do not need it. The carrier is cool to be used in other kind of experiments like RNA-IP.
Anyhow, if you really like to use the carrier, you must be aware that you will get it always during purification. RNA micro-spin columns are not size-exclusion columns, but ionic-exchangers. The purification and cleaning process by the column is based on the selective binding of negative molecules (RNA) to a anion-exchange resin (embebed in the filter of the spin-column) under slightly hydrophobic conditions (you must add ethanol or isopropanol to your sample before loading the column). So, you have no way to get rid of the MS2 RNA by using this protocol.
Your results are probably due to some technical artifact, but in principle the MS2 RNA should also bind to the purification micro-spin column. Be sure that you performed the protocol exactly in the same way that you did it for plasma, but substituting the plasma by your PBS-MS2 solution.
I would advice you to give a try without the carrier. And do not forget that using Nanodrop to quantify RNA coming from plasma samples is useless because of the sensitivity thresold of the machine. You need to use more sensitive devices as Qubit or Bioanalyzer.
Best of luck.
Paco
Answer
Hi Mingliang
I agree with previous answer about the homology of known lncRNAs. However do not be dissapointed if you did not find anything homologous to your specific lncRNA, since those guys have the tendency to be very specific of group and species. I am not a plant person, but in animals it is really amazing how these guys are so specific from genetically defined groups.
Whatever the case, if you have coding expression data, another possible strategy to guess the function of lncRNAs is to check for the genes that are close together with it within the genome (let's say around 100 kb to your lncRNA) and see if they are UP or DOWN compared to your lncRNA. This could pinpoint cis-regulatory effects (or at least guess). Of course that you will always miss the trans-regulatory effects exerted far away in the genome.
In conclusion.. it is very difficult to guess the function of a lncRNA either in plants or animals just by its sequence. The best way would be to knock-down the gene (by CRISPR or LNA gapmers), and see phenotypic effects if possible.
Good luck. Best
Paco
Question
Hi there
I am dealing with a long list of miRNAs... I would like to get some tool to organize them by functional groups, clusters, chromosome location, tissue expression, etc...
Any suggestion?. Thanks in advance
Best
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Hi there
I did not solve anything. I gave up
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Hi Hawking
Still not.. I wrote an email, but no answer. Still waiting.
Hope you have best luck
Answer
Hi there
Just to make some more noise about... I have found this application, decodeRNA, which uses an integrative approach for decoding miRNA and lncRNA function. Unfortunately you just can use single miRNAs, but I think that it could help.
You can use also miRNet, which is devoted for miRNAs, and in this case you can analyze the action of multiple miRNAs over cohort of genes and pathways. This app only work with validated miRNA targets.
Keep moving !.. Best
Paco